Das H K, McPherson J, Bruns G A, Karathanasis S K, Breslow J L
J Biol Chem. 1985 May 25;260(10):6240-7.
The human apo-E gene has been isolated from a lambda phage library using as a probe the previously reported apo-E cDNA clone pE-301. Lambda apo-E was mapped and subcloned, and the apo-E gene was completely sequenced. The DNA sequence was compared with that of a near full length cDNA clone pE-368 and revealed three introns. The first intron was in the region that corresponds to the 5' untranslated region of apo-E mRNA. The second intron interrupted the codon specifying amino acid -4 of the apo-E signal peptide. The third intron interrupted the codon specifying amino acid 61 of the mature protein. Analysis of the DNA sequence revealed four Alu sequences. Two were in opposite orientations in the second intron, and one each occurred in the regions 5' and 3' to the apo-E gene. There were two base differences between the apo-E gene sequence and the sequence derived from the cDNA clones. At the codon for amino acid residue 112, the apo-E gene contained CGC, specifying Arg, whereas the cDNA contained TGC, specifying Cys. The other base difference was in the area corresponding to the 5' untranslated region of apo-E mRNA. Apo-E is commonly polymorphic in the population and the data suggest that the genomic clone was derived from the epsilon 4 apo-E allele, whereas the cDNA clones were derived from the epsilon 3 apo-E allele. S1 nuclease protection and primer extension experiments allowed the tentative assignment of the cap site of apo-E mRNA to the A approximately 44 base pairs upstream of the GT that begins the first intron. The sequence TATAATT was identified beginning 33 base pairs upstream of the proposed cap site and is presumably one element of the apo-E promoter. Finally, the apo-E gene was mapped in the human genome to chromosome 19 through the use of DNA probes and human-rodent somatic cell hybrids.
已使用先前报道的载脂蛋白E（apo-E）cDNA克隆pE-301作为探针，从λ噬菌体文库中分离出人类apo-E基因。对λ载脂蛋白E进行了图谱绘制和亚克隆，并对apo-E基因进行了全序列测定。将该DNA序列与一个近乎全长的cDNA克隆pE-368的序列进行比较，发现了三个内含子。第一个内含子位于与apo-E mRNA的5'非翻译区相对应的区域。第二个内含子打断了编码apo-E信号肽第-4位氨基酸的密码子。第三个内含子打断了编码成熟蛋白第61位氨基酸的密码子。对DNA序列的分析揭示了四个Alu序列。两个在第二个内含子中呈相反方向，另外两个分别出现在apo-E基因的5'和3'区域。apo-E基因序列与来自cDNA克隆的序列之间存在两个碱基差异。在氨基酸残基112的密码子处，apo-E基因含有CGC，编码精氨酸，而cDNA含有TGC，编码半胱氨酸。另一个碱基差异位于与apo-E mRNA的5'非翻译区相对应的区域。apo-E在人群中通常具有多态性，数据表明基因组克隆源自ε4 apo-E等位基因，而cDNA克隆源自ε3 apo-E等位基因。S1核酸酶保护和引物延伸实验初步确定了apo-E mRNA的帽位点位于第一个内含子起始的GT上游约44个碱基对处的A。在拟议的帽位点上游33个碱基对处开始鉴定出序列TATAATT，推测它是apo-E启动子的一个元件。最后，通过使用DNA探针和人-啮齿动物体细胞杂种，将apo-E基因定位在人类基因组的19号染色体上。
